Multilineage hematopoietic growth factor interleukin 3 and direct activators of protein kinase C stimulate phosphorylation of common substrates.

In order to investigate early signal transduction events in myeloid cells, the phosphosubstrates of an interleukin 3 (IL 3)-dependent cell line, FDC-P1, have been analyzed. Using synthetic diacylglycerol as a direct activator of the unique calcium-phospholipid-dependent phosphotransferase protein kinase C (PK-C) and genetically engineered homogeneous IL 3, we have demonstrated a common element to signal transduction events associated with these stimulants. One novel substrate, p68 (68,000 kd), was rapidly phosphorylated in either IL 3- or diacylglycerol-stimulated cells. The phosphorylation of p68 was dose-dependent, with both the physiological ligand and diacylglycerol inducing the same maximal level of phosphorylation. Phosphorylation of p68 occurred in a time-dependent manner analogous to previously described kinetics of PK-C subcellular redistribution in the FDC-P1 cell line. The p68 substrate was also phosphorylated in a cell-free system under conditions designed to activate PK-C. Phosphoamino acid analysis demonstrated that the p68 molecule phosphorylated in intact cells as well as in a calcium-phospho-lipid-dependent cell-free system was phosphorylated on threonine residues, not tyrosine. These data support the hypothesis that the activation of PK-C that occurs after IL 3-receptor interaction which leads to the rapid phosphorylation of cellular proteins is an important element of the signal transduction mechanism in FDC-P1 cells. We propose that phosphorylation of the p68 molecule is a physiochemical marker for the activation of PK-C in myeloid cells, in response to the growth-promoting physiological ligand.